Bottom Line:
Due to its inherent resistance to the major antibiotic classes and its facultative intracellular pathogenicity, an effective vaccine would be extremely desirable, along with appropriate prevention and therapeutic management.All three epitopes were shown to be immunoreactive against human IgG antibodies and to elicit cytokine production from human peripheral blood mononuclear cells.Furthermore, two of the peptides (F51-69 and F270-288) were found to be dominant immunoreactive epitopes, and their antibodies enhanced the bactericidal activities of purified human neutrophils.

Affiliation: The Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.

ABSTRACTBurkholderia pseudomallei is a Gram-negative bacterium responsible for melioidosis, a serious and often fatal infectious disease that is poorly controlled by existing treatments. Due to its inherent resistance to the major antibiotic classes and its facultative intracellular pathogenicity, an effective vaccine would be extremely desirable, along with appropriate prevention and therapeutic management. One of the main subunit vaccine candidates is flagellin of Burkholderia pseudomallei (FliCBp). Here, we present the high resolution crystal structure of FliCBp and report the synthesis and characterization of three peptides predicted to be both B and T cell FliCBp epitopes, by both structure-based in silico methods, and sequence-based epitope prediction tools. All three epitopes were shown to be immunoreactive against human IgG antibodies and to elicit cytokine production from human peripheral blood mononuclear cells. Furthermore, two of the peptides (F51-69 and F270-288) were found to be dominant immunoreactive epitopes, and their antibodies enhanced the bactericidal activities of purified human neutrophils. The epitopes derived from this study may represent potential melioidosis vaccine components.

pntd.0003917.g006: Domain organization and 3D crystal structure of FliCBp.Schematic secondary structure ribbon representation of the crystal structure of FliCBp. The N- and C-termini are labeled as are the diverse secondary structural features with α-helices in cyan and β-strands in magenta. Peptides 96–111, 213–233 and 270–288 are highlighted in red, green and black, respectively. This figure was produced using MacPymol.

Mentions:
Flagellins vary in dimension (28–65 kDa) and contain at least two essential highly conserved domains, D0 and D1 that mediate assembly of the flagellar filament, and may contain a second (D2) or third (D3) variable domain. Typically, removal of the D0 domain is necessary for successful crystallization. This was not the case for FliCBp where, however, the first 44 N-terminal residues (corresponding to the D0 domain) are not visible in the electron density map because of structural disorder. Overall, FliCBp adopts the canonical flagellin fold and presents two domains, a conserved D1 domain (residues 69–167, 287–326), and a variable D2 domain (168 and 286) (Fig 6). D1 forms a helical rod (α1, α2 and α5) involving residues contributed by both the N-terminus (residues 69–170) and C-terminus (residues 285–326), a β-hairpin domain (β1-β2) and a 310 α-helix (η1). Domain D2 (residues 172–287) contains a three-stranded antiparallel β-sheet, two α−helices (α3 and α4) and two 310 α-helices (η2 and η3). Domain D2 is connected to D1 via two anti-parallel regions that house two short 310 α-helices (η1 and η3) that interact with each other, thus stabilizing the relative positions of the two domains (Fig 6).

pntd.0003917.g006: Domain organization and 3D crystal structure of FliCBp.Schematic secondary structure ribbon representation of the crystal structure of FliCBp. The N- and C-termini are labeled as are the diverse secondary structural features with α-helices in cyan and β-strands in magenta. Peptides 96–111, 213–233 and 270–288 are highlighted in red, green and black, respectively. This figure was produced using MacPymol.

Mentions:
Flagellins vary in dimension (28–65 kDa) and contain at least two essential highly conserved domains, D0 and D1 that mediate assembly of the flagellar filament, and may contain a second (D2) or third (D3) variable domain. Typically, removal of the D0 domain is necessary for successful crystallization. This was not the case for FliCBp where, however, the first 44 N-terminal residues (corresponding to the D0 domain) are not visible in the electron density map because of structural disorder. Overall, FliCBp adopts the canonical flagellin fold and presents two domains, a conserved D1 domain (residues 69–167, 287–326), and a variable D2 domain (168 and 286) (Fig 6). D1 forms a helical rod (α1, α2 and α5) involving residues contributed by both the N-terminus (residues 69–170) and C-terminus (residues 285–326), a β-hairpin domain (β1-β2) and a 310 α-helix (η1). Domain D2 (residues 172–287) contains a three-stranded antiparallel β-sheet, two α−helices (α3 and α4) and two 310 α-helices (η2 and η3). Domain D2 is connected to D1 via two anti-parallel regions that house two short 310 α-helices (η1 and η3) that interact with each other, thus stabilizing the relative positions of the two domains (Fig 6).

Bottom Line:
Due to its inherent resistance to the major antibiotic classes and its facultative intracellular pathogenicity, an effective vaccine would be extremely desirable, along with appropriate prevention and therapeutic management.All three epitopes were shown to be immunoreactive against human IgG antibodies and to elicit cytokine production from human peripheral blood mononuclear cells.Furthermore, two of the peptides (F51-69 and F270-288) were found to be dominant immunoreactive epitopes, and their antibodies enhanced the bactericidal activities of purified human neutrophils.

Affiliation:
The Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.

ABSTRACTBurkholderia pseudomallei is a Gram-negative bacterium responsible for melioidosis, a serious and often fatal infectious disease that is poorly controlled by existing treatments. Due to its inherent resistance to the major antibiotic classes and its facultative intracellular pathogenicity, an effective vaccine would be extremely desirable, along with appropriate prevention and therapeutic management. One of the main subunit vaccine candidates is flagellin of Burkholderia pseudomallei (FliCBp). Here, we present the high resolution crystal structure of FliCBp and report the synthesis and characterization of three peptides predicted to be both B and T cell FliCBp epitopes, by both structure-based in silico methods, and sequence-based epitope prediction tools. All three epitopes were shown to be immunoreactive against human IgG antibodies and to elicit cytokine production from human peripheral blood mononuclear cells. Furthermore, two of the peptides (F51-69 and F270-288) were found to be dominant immunoreactive epitopes, and their antibodies enhanced the bactericidal activities of purified human neutrophils. The epitopes derived from this study may represent potential melioidosis vaccine components.